Supplementary Materials for

Modulation of DNA methylation influences cartilage formation in murine chondrogenic models

Judit Vágó, Katalin Kiss, Edina Karanyicz, Roland Takács, Csaba Matta, Tibor Rauch, Róza Zákány*

#Corresponding author. E-mail: [email protected]

This PDF file includes:

 Sequences of primer pairs used for the PCR assay analyses.  Sequences of primer pairs used for the RT-qPCR reactions.  Sequence data of the 3’UTR regions of Dnmt3a, Ogt and Tet1 genes with insert flanking T7 promoters for antisense probe preparation. These regions were cloned into pDrive vector and later amplified for digoxigenin-labelled RNA probe preparation.

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Epig.Modif. Gene Forward Reverse Dnmt1 CAGAGGAGAGAGACCAGGATAA GCTGTTACCTCTTCCAGTTTCT Dnmt3a CAAGGGACTTTATGAGGGTACTG TTCTCAAAGAGCCAGAAGAAGG Dnmt3b GAACCCTGGAGCTGCTATATG CAACTTGGGTGGCTCAAATTC Tet1 CCAAGTGGGTGATCAGAAGAA CACAGCAGGATAAGGACAACTA Tet2 AGGATGCAATCCAGACAAAGA GCTTTACCCTTCTCTCCATACC

DNA methylationDNA Tet3 GAACTCATGGAGGATCGGTATG CAGTGTGTGTCTTCGGATCA Ogt GACTGGCGACTACACAGATTA GGGAACGGGTGGTTACAATA Atf2 ATGGCAGTGGATTGGTTAGG GAGAAGCCGGAGTTTCTGTAG Crebbp CAACAAACCATCCTGGGATCT GGGTCTATGGGATTTGGGTTAC

Ep300 GGCAATGCTGGCAGTTTATT TTCCCATCTTAAGTGGTTGGG Hat1 GCCTGAAGATCTTGCTGTACTA TCATCTGCCTCCACACAATC Kat2a GCTCTTGGGAATGGTAGTAGATG GCAAGAGCTTGAAGAGGTAGAA Kat2b GAGGAGACCTCCAGCAGATAAT TGAGAAACGTGAGCAGCAAG Kat5 TTTCCCTCAGAATGGGTCAG CATCAGTGCCCAAGCAATTAG Histone acetylation Kat6a CCCATCTGTAGCTTCTGTCTTG CACGATGGATGACCGCTATT Kat6b GTGCCGATCCCATTCCAATA ACAGGATGGGTGTCCACTA Kat8 TCTATGTTCACTATGTGGGCTTTA GCCAGCTCACTCAGGTATTT Hdac1 GATGCAGAGATTCAATGTTGGTG GATGTCCGTCTGCTGCTTATTA Hdac2 CCTCATAAAGCCACTGCTGAA CCGACGTTAAATCTCTGCATCT Hdac3 TGATGACTGCCCAGTGTTTC GGCCCAGTTGATGGCAATA Hdac4 CTTGAGGGCCGCTTTATCA GGAAAGAAACACAACCAAGTCTATC Hdac5 AGGAGGAGCTGGAGAAACA GATGGCACTCTCTTTGCTCTT Hdac6 CCCAATCTAGCGGAGGTAAAG GTTCAGATCCAGCCCTTGAA Hdac7 CAGAAGCTGGCTGAAGTGAT GCTCAAGAGTTCTGTAGGGAATAC Hdac8 CCTTCCACACTGATGCCTATC GGCAGTCATAACCTAGTCCATATT Hdac9 GGAGCACATCAAGGAACTTCTA GCCTCTCTACTTCCTGTTCTTG Hdac10 AGACCCAGACCCTGGATAAA AAAGGTGTCCGGGTGAAAG Hdac11 GAGCTGAAGTGGTCCTTTGT TCCTCTGCACAAGGAAGTTG

Histone deacetylation Sirt1 GGATCCTTCAGTGTCATGGTT CACCGAGGAACTACCTGATTAAA Sirt2 CCAACCATCTGCCACTACTT CGTGTCTATGTTCTGCGTGTA Sirt3 GCCCAATGTCACTCACTACT GATCCCAGATGCTCTCTCAAG Sirt4 CGCTGCTCAAGATCCCTAAG GGGCAGCTCTCATTTCTGTAA Sirt5 GTGTCTAGTGGTGGGAACATC GGGTGGTCTCCATGTTAAACT Sirt6 CCCAAGTGTAAGACGCAGTA CAGTCCAGAATGGTGTCTCTC Sirt7 GTGGTGTCTCAGAACTGTGATG CAGGAGGTGCAGACTTCAATATAC

Table S1. Sequences of primer pairs used for the PCR assay analyses. The table continues on the next page.

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Ash1l GAGAGAGGAACTTCGTGCTAAA TGTACAGCGATGCTGAGTG Dot1l CATCACTACGGAGTGGAGAAAG ACCTCGTTCCAGTGTGTATTC Ehmt1 GTCTGACCTGAGTTCTGAATCC CTGCTCGGCTTCTTTCTACTT Ehmt2 ACAACGCACGCCACTAAT CATCCTCTTCCTTGCTGTAGAC Ezh1 GGAGCAAAGGCTCTGTATGT GGCTGGACACGAAGTTTCT Ezh2 CATCCCGTTAAAGACCCTGAA ACAGTTTCGTCTTCCACCATAA Fbxo11 GATGGATTTGCTGCAGGTATTG GCCTCCAAATCGGTTGTTAAAG Kmt2a CCAGACCCTCCTGTTCTTACT CGCTGAACTCCAACACAGATAC Kmt2b GCTGGACTGGAAAGCAGAA CTAGGTCGCAATCCTCCTTAAA

Kmt2c TGTTAAGGCCTCACACCTTG GGAGTGGACAAACTGCTTACT Kmt2d GAGACATGTGACAAAGGGTATCA CATAGCCGGCATGTCTTACA Kmt2e AGCCCAGGAGTTTGGATAAAG AGGAACTTCATCACCACTTTCA Prdm4 GACACACCAATAGAGAGCAGAG ATGGAAGGACACCAACAACA Prmt3 AGACTTAAGAATCCTACGGTTGAA GGAGTAGACACAGGCTCATAAAG

Histone methylation Prmt4 (Carm1) GGCGATTTGCACAGGATAGA GGAGCCAATGAAAGCAACATC Prmt6 GGTGGAACAAGATACGGACAT CTCATGGTCTCCCACTTTGTAG Setdb1 ATCAGCTCAAGATGTCCAGAAG CCTGACCCAAGGTTCCTTTAT Setdb2 TCTGCCACAAATGGAGACTATG CACTCTGAGTCACAGGTGTATG Smyd1 GGAAGTGGTGAAGGAGATGATAC GCTTCACAACCTCGTGATACA Smyd2 CGGCGATATTCCCTGATGTT TCCTGTACAGCTCTGACTTCT Suv39h1 CTCTGCATCTTCCGCACTAAT CTGAGGTAATAATCTCTCCCACATAC Suv39h2 GTGAATCATAGTTGTGACCCAAATC CAGCTCTTCTCCAGCGTTTAT Suv420h2 CTGCTCTCAAGACCCACATTT AAGCACGGGTAGACACAATC Jmjd1c GCCATGATGCCAACAAGATATG GTACAAAGAACCCTGGCAAATG Kdm6b GAGGAACCAGACAGCACTAC CTTCCACCTCTTGGCATCA Jmjd4 AAGTATGGAGACGCGGTTG GTAACTGATATAGTCGCGGAAGG

Jmjd6 CCATTGCCATCACCCAGAA GCTCCTGTTTCAAGATCCTATACC Jmjd7 CTTGCACAAGGACCACTATGA TGCTGGTGTGTAGAGATTGTAAG Jmjd8 GAATTGCAGGAGCTGGATCT TGTCTTCTCAGGAGGGTAGAG Kdm1a TGGGATCAGGATGATGACTTTG GCACTGCTGTGTTCAGTTTAAT Kdm1b GACTCAAATCTCCAGTGCAAAG GATGGCACCTCTCTGTAGTATG Kdm3a TCATCCACAGCAAAGACAGAA GGCCTCTTGGATGAATCAGAA

Histone demethylation Kdm3b GTGACCAAGCAGAGGATTCA TTGCTCTTCTCCAACCTTCC Kdm4b CAGCCGGATTACACAGGAAG TGGACTCAGCGCAGTTAAAG Kdm4c AGTTGTTATGGTGTCCCTTCTC GAGCCCACTGATTGTTCTTAGT Kdm5a CCTGAATGAACTTGAGGCAATG CACCACAGGGATCTTCAATGTA Acan TGGAGGTCATAGTGAAAGGTATTG ATGATGGCGCTGTTCTGTAG Col2a1 TGGCTTCCACTTCAGCTATG GGTAGGCGATGCTGTTCTT

Prg4 TGGAGGACTAACAGGGAAGATA CTGAATGTTGCCACCTCTCT Alpl GCCATGACATCCCAGAAAGA GCCAGACCAAAGATGGAGTT

ogenesis r Col1a1 ACAAGGTGACAGAGGCATAAA ACCAGGAGAACCAGGAGAA Col10a1 AACGCCCACAGGCATAAA CTCCTCTTACTGGAATCCCTTTAC

Chond Spp1 CTTTCACTCCAATCGTCCCTAC TGGCATCAGGATACTGTTCATC Znf219 CGGCCACCCAAGAAGAAA CGCCGAGCGGAAAGATT Table S1. Sequences of primer pairs used for the PCR assay analyses. 3

Gene Accession number Primer sequence Amplicon size

1. Epigenetic marker genes Dnmt3a XM_006514953.3 CTGTCCCTACGACCAGTGC 110 CTGATGTCAAGCCCTCGGAA Tet1 NM_001253857.2 CTGGGGCCATCCAAGTCAAT 120 TGTGTGAACCTGATTTATTGTGGT Ogt NM_139144.4 TTCAGTATTCTGTGCCGCCC 188 TCGTTGGTTCTGTACTGTCGG 2. Chondrogenic marker genes Sox9 NM_011448.4 GGAAGTCGGTGAAGAACGGA 158 AGATTGCCCAGAGTGCTCG Col2a1 NM_031163.3 TCATCTTGCCGCATCTGTGT 164 TGCCCCTTTGGCCCTAATTT Acan XM_006540566.1 ATTCCCGCCCACCTACCT 190 GCTGACTAGGTTTCGGAGCA 3. Reference genes Actb NM_007393.5 AGATCAAGATCATTGCTCCTCCT 174 ACGCAGCTCAGTAACAGTCC Sdha NM_023281.1 TCGACAGGGGAATGGTTTGG 110 GGACTCCTTCCGAGCTTCTG

Table S2. Sequences of primer pairs used for the quantitative real-time PCR analyses. Nucleotide sequences, gene accession numbers and amplicon sizes are displayed for each primer pair.

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>T7promoter-Dnmt3a (anti-sense) Clone#:D7 chr12:3,907,819-3,908,735 GCCAAGCTCTAATACGACTCACTATAGGGAAAGCTCGGTACCACGCATGCAGACGCGTTACGTATCGGATCCAGAATTCAGATTGCACG CGAGTCTGGATAAATTCCACACTCAGCTTCCCCAGGGTCCGACCACCTCAGCTGAGCCCCTTTTTGGCAGGTGAAGCTAGGCATGGGGC AGGGGTGGGGGGTGATATGGGTCATCCTTCAGTTTGTTAAAACCCCCTCCAGCTGCTCCCCTCTCCCCATCCTGGGCTGAAACCCTTTG CACAGAGGGCCAGGCGGAAGCTGATGTCTTTGCTCATCTCGCTGTTTTGAAAGCACCATTATGAAGTCGGGATGTACAGTAGTTAACAG TACCTTTTATATATATATATCTCATATCTATCATATATATATAAACTGTAAACAAGAGGTAACAGCGGCTTCTAGAAACTGGTTTTCTT CAAGGTTTCCTGTGTGGTAGGCACCTGAAATACTGTAGAAAAGTGTCTGGTGTGGTGTTCTCTCAGCGCCCTGCTGCTAGTTGGGTTCT GTTTTGCATGACCAGGAATGGTGCTGTTGAGACTGCTCGGGGGTGGGAGAGGAGACTCTGCCTCCGAGGGTCTCTATTCTCTTCCTGTT TGTGCTCCTATCTGATCAGGCTAGAGACAACCAAAGAGATATATAGAAAAGGAAAAAAAAAAAACCAATATTTTAAGTTTTTATAGAAT TTTCCCTCTCTCCCTTTCCTCTCCAAGGCATTTCTACTTTTTGTTGTTACTGATTTTGCTGCAATAACTCTCTTGTTTCAGTCACAGCA AGAAACAAAAACCCAAACAAACAGAAAACCCCTCTGAAAAAGAGTAGAAAACAAAAAGTTCGCCTCCTAGGACCGGAGGGGAAGAAGGG GAGGGGCCTATTTTGCTTTTTAGATAGGACTGAAGGTTAACATTGAAAACTCAGGAGATGATGTCCAACCCTTTTGCAAGGCCAAGCCC TCCGGCGTTTTCCTCCTCTGGGGTTTGCTTTAAATTCCTTTTTCTCTTCTGGGTGCTGAACTTTTCTCCGTCCTCTCGTTCTTGGGGGT TTAATGGTTTTGTTTTGTTTTTGGTTTGTTTGTTGGTTAAATTTTTTTATCATCACTACTTCAGTTTGCCCCCATGTCCCTTAACCACA AATCACGAATTCGTCGAAAAGCTTCTCAAGCCTAGGCTAGCTCTAAACCCAACGTGTGGGGGCCCCAACTCCCGGCCCCTTGAATTCTA TAAGGTCACCTAAAGGGCCGCACAATTCCATGGGCCGCCGTTTTACAACGCCGGGACTGGAAAAACCCTGGGGTTACCCAATTT

>T7promoter-Ogt (anti-sense) Clone#:O2 chrX:101,682,485-101,683,489 CCAAGCTCTAATACGACTCACTATAGGGAAAGCTCGGTACCACGCATGCAGACGCGTTACGTATCGGATCCAGAATTCGTGATTCTACC AAGGCCGAATCCATAGAGTTTGAAGCTAAGTCCAGACATTCTAACACAGTCTAAAAATAAAATAACTGCCTGTATAGAGATGCTTCTTC AGAGCGGTGAACACCATTTAAGGTACTGGAGAGAACTGTAATTTACTAACCAAACATTTGAATACTTATATACTTACCTGGGATTTCAT TTCTGCTGAAAGAAATGGGAAGAACAGGACTCACTTTAAAAAAAAAAACAAACTTTAGAAAGGAAGGTAAAAATCTTATCACACTATTA CTTTTGGAAGCAGCATAGGAGGGGCAGTCAAGGGCTGAAACACTGGTGAAACAGGTAAAACTTAGGCAGAAAAAAACCCTATATTATTA TCAATAACAGTACCACAACTGTGACCTTCATGATTAATATTCACTCCTAAGAATTTTCATTTGGCACCAGATGGCATGTTCGAGAGAAA CACCCTGGGATGTCTGAAAGCAGACATGGGTTTGTTAGCAGTCACAGGAGAATTTAACGGGAGCTGGGGTGTGTAAAACAGGCTGAAAA GTCTTAAGTCATCATCTAAACAAAGCACTGACAGTGCCAAGCATTAGGTCAGACACTAAAATGACTGATATAGGCTCATGTGGTTTATA AAACCTATAAAAAGACTACACCAGCAAGGTCCCTGTCACCCTGTCAGAGTTCAGACACTAAAACAGGAAGCAACATTTTAGTTTAAAAC CTCAGAAGAATCAAGATCACTTCACATGAGTAAAAATACCTGAAAGCAAACATTTTTAAAAGCTCCAAAACCCAAAATATAAAGAAAAA AAAAAGGATCAGAAGACTTGATCAATCCACGGCATCACAAAACGGCTGGTTAATCTCTATCTCCCCTCCACATTAAGCATCCCATGGAA GACCAAGGGAGACCAGACCTTCCAGACCTGTGCACCACCTGTTTGCTGCAAAACTCTAAGAGATTCCTTGTGGAATAGCATTTCTTCTT TCTTGTCTCTCCCTATCATGCAGAATCTGAATTCGTCGACAAGCTTCTCGAGCCTAGGCTAGCTCTAGACCACACGTGTGGGGGGCCCC AACTCCCCGGCCGCTGTATTCCATAGGGGCCACCTAAATGGGCCGCACAATTCACTGG

>T7 prom-Tet1 (anti-sense), Clone#T1 chr10:62,811,427-62,812,547 CCAAGCTCTAATACGACTCACTATAGGGAAAGCTCGGTACCACGCATGCAGACGCGTTACGTATCGGATCCAGAATTCGTGATTGGGTA AGGGTGACAATGCTAACGCAAGCATGTCCCCTCAATTATCTCTATAGAATATTTCCTTCTCATTTGGGAAGGACATGTCTCTTAATTAT GTATTTTCTAATCTTGAAACACTGGGAACTTTTGACTGATCCCAATTTGCCTCCTTTTCCTTTGAAAACACCTGTAATTGGGTCATATA GTTAATTGACGAGGTGACTTCAGAAGACAATATTTTTTCTAGTTAAAGAGGGAAAATTGATATTGGTAGCATCTTGTATTTAAATTTTT TTTTTTAGAGACCCTCTATTCTATAAGTAAAGAACAGAAGCCGTGTGTTATTTGAGTACAGGCTGAGGTTAACGGGATGGGACTCTACA GAGCAAGAACAAATGACTTCTCTGGTATTCAAATATGCACACATAATAGACAATAGGATTCTGCTACTATACTATTTCAAGTTACTCTT AAAAGTAGAAAGTGCTTTTTAAAATATATACATTTATAGTTAGCGAACAGCTTCCAACCATCATGAGAATAGAGAACTACTTTATATAG GAGAAATGTTTTAAGAGAAAAAGCATTTCATTGGTTTACTTCAAAGAAATGTGCAGACACGATGGCAATGATTGGAAGAGGCTTGAGCA ATGGCACCAGAAAACAATCACTCAGCTCATCACTCCGTGTGTTGATGTGGTTAAAGCACCAGGAACTGTGTAGAGTTAACACTACATCT TGGTCTTTACTTCTTATTGCAGGAACCTGTTATAAACCCTCTGAATAGTAAACAAACCCGGGGGAAAGCACCAGACTCTATGGTTCTAG CATGGCCAACACCTAACCAAGATGTTCCTTTAAGTATTTAAAACTATAAAAAGTTTATTTTTGCTGAAAATGTTCACCTTCTACCAATT CTCAAAATGTAATCACAGTTAAGTATTCACCCTTTTTTTTTTTTTTTTAATATTTACGTCGGAATTGAAATGGGCGAAACAAAGCTTCC GTAAACAACAAAATGACTTTCTTTTTAACAGCACCGGAAAAAACAAAAAAACCAAAGAAAACCAACCAACCAAACAAAAATTCCCCCTT ACCCCCCTTTAGCCAAGGGCTTTACTTGGAGAAAAGCCTTTAGACCCACCGATTGGAAGGGAATCTGAATTCGTCCAAAAACTTTCCCA AGCCTAGGCTAACTCTAAAACCAACCGTGGGGGGGGCCCCAACTTCCGGGCCCCTTGAAATCCTTAAGGGGCACCCTAAAGGGCCGCCC AAATTTCCCGGGGCCCGCCTTTTTACAAGGCCCGGGACTGGGAAAAACCTGGGGGGTTACCAAATTAAAACCCCTTGGCGAAAAATCCC CCTTTTCCCCCGTGGGGCGTATAAAGACAAGAGG

Table S3. Sequence data of the 3’UTR regions of Dnmt3a, Ogt and Tet1 genes with insert flanking T7 promoters for antisense probe preparation. These regions were cloned into pDrive vector and later amplified for digoxigenin-labelled RNA probe preparation.

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